Research Description
In my laboratory, we are interested in how oxygen is produced by photosystem II (PSII) of higher plants. PSII contains a cluster of manganese ions at the site where O2 is produced from H2O. We are particularly interested in how chloride functions to activate catalysis in this system. To carry out this research, we combine biochemical techniques, such as protein purification and enzyme kinetics assays, with physical techniques, particularly electron paramagnetic resonance spectroscopic. Projects in our lab have been funded by Research Corporation, the Dreyfus Foundation, and the National Science Foundation.
Photosystem II
Higher plants and many other photosynthetic organisms evolve oxygen during the conversion of light energy into chemical energy. Although O2 is a byproduct of this process in plants, the presence of O2 in the atmosphere is of essential importance for the survival of animals and other respiring organisms. Because it is needed by all respiring life forms, its formation is among the most important biochemical reactions for the support of life on earth. In addition, the production of O2 by plants is the first step in the process that converts solar energy into a chemical form usable by all living things. Photosystem II, where O2 is produced, is a large protein complex that is embedded in the thylakoid membrane network within chloroplasts. In addition to the manganese cluster, the oxygen evolving complex (OEC) contains calcium and chloride ions. Although much is known about photosystem II at a molecular level, the mechanism by which water is converted into oxygen is poorly understood and the roles of Ca2+ and Cl- are not clear.
The manganese cluster cycles through a series of oxidation states called S-states, as described by the Kok model. They are designated S0 through S4, where S0 is the lowest and S4 the highest oxidation state. These S-states are probably made up of 4 manganese ions in varying ratios of Mn3+ and Mn4+. For example, the S0 state may correspond to the oxidation state (Mn3+)3Mn4+ and the S1 state to the state (Mn3+)2(Mn4+)2 one step up. Progression from one state to the next requires the absorption of a unit of photon energy by the chlorophyll of the photosystem II reaction center, P680. Oxygen is evolved upon reaching the highest oxidation state S4, which is unstable, and the system returns to the lowest oxidation state S0 to begin the next cycle. A tyrosine residue near the manganese cluster, known as Tyr Z, accepts electrons from the cluster during the oxidation cycle, and passes them on to the reaction center. Chloride and calcium ions are required as cofactors for this process and their participation is regulated in higher plants by extrinsic PSII protein subunits PsbP and PsbQ, with molecular weights 23 and 17 kDa, respectively. The Ca2+ ion is now believed to be bound as a part of the Mn cluster, with the formula Mn4Ca. One Cl- ion binds near the oxygen evolving complex with very high affinity and is involved in the catalysis of oxygen formation. The Mn cluster cannot proceed past the S2 state without Cl-.
A possible version of the S-state (Kok) cycle of the OEC
Anion activators and inhibitors of O2 evolution
We are interested in how Cl- functions in oxygen evolution because it appears to be needed for some specific step in the catalysis of O2 formation. An understanding of its function would help in understanding of the overall catalytic mechanism. Understanding how Cl- functions in PSII would also help to understand its role in other Cl--binding enzymes, such as cytochrome oxidase. One way to understand how chloride functions is to replace it with other anions. The chemical properties of the various substituting anions and their effects on catalysis can reveal what is required for normal function.
We have carried out studies using anions that function as activators in place of Cl- and anions that act as inhibitors. Other anions besides Cl- that can activate oxygen evolution include Br-, NO3-, and I-. Br- can function almost as well as Cl-, while NO3- and I- are not as effective. The effect of I- is complicated because it is also an inhibitor at higher concentrations. The effectiveness of the activators can be measured using steady state kinetics as described by the Michaelis-Menten equation, where Vmax is the maximum velocity of catalysis obtained at high concentrations of activator and KM gives the concentration of activator required for half maximal velocity. The experiment is carried out by measuring the rates of O2 evolution using an O2 sensitive electrode for various concentrations of the activator.
We have also characterized the effects of anion inhibitors using enzyme kinetics methods. Some of the inhibitors we have studied include F-, N3-, and I-. Inhibitors F- and N3- are competitive with Cl-, which means they prevent the Cl- from binding to its site of activation. These inhibitors tend to have weak base properties. In contrast, I- inhibits from a different site by binding after the activating Cl- binds (uncompetitive inhibition). This second site is evidently near the OEC. These studies are carried out by measuring the rate of O2 evolution for various concentrations of both inhibitor and Cl- and analyzing the data using standard steady state kinetics methods for inhibitors.
EPR spectroscopy
Our research also involves the application of electron paramagnetic resonance (EPR) spectroscopy to the study of photosystem II. EPR spectroscopy depends on the presence of unpaired electrons and observes transitions between the energy levels of the spin states of these electrons. Two major types of biological centers can be studied with this technique: organic radicals, which often involve aromatic ring systems, and paramagnetic transition metal complexes, involving ions such as Fe3+, Cu2+, and Mn2+. EPR spectroscopy is ideal for studying electron transfer processes, such as those that take place in photosystem II, because the sites of electron transfer often involve the formation of organic radicals or changes in metal oxidation states.
EPR experiments in our laboratory focus on signals that are produced from the manganese cluster where oxygen is produced or the tyrosine radical, Tyr Z, that is nearby. The different oxidation states of the Mn cluster show different EPR signals. The S2 state shows two well-known signals: a multiline signal, which shows multiple splittings caused by the nuclear spin of Mn; and a broad low field signal (called the “g=4.1 signal”), which originates from an S=5/2 spin state. The presence of Cl- and other anions has a strong effect on these signals. For example, the S2 state multiline signal does not form in the absence of Cl- and the g=4.1 signal is modified by the presence of F- or N3-. These effects indicate that these anions have a direct influence on the Mn cluster. More recently we have become interested in understanding the effects of anions on a signals from the S1 state and the S3 state.
Selected and Recent Publications by Dr. Haddy
“Characteristics of Iodide Activation and Inhibition of Oxygen Evolution by Photosystem II” David I. Bryson, Ninad Doctor, Rachelle Johnson, Sergei Baranov, and Alice Haddy (2005) Biochemistry, accepted
“Q-band EPR of the S2 state of Photosystem II confirms an S=5/2 origin of the X-band g=4.1 signal” Alice Haddy, K. V. Lakshmi, Gary W. Brudvig, and Harry A. Frank (2004) Biophysical Journal 87, 2885-2896
“Converting a Maltose Receptor into a Nascent Binuclear Copper Oxygenase by Computational Design” David E. Benson, Alice E. Haddy, and Homme W. Hellinga (2002) Biochemistry 41, 3262-3269
“Using a Molecular Modeling Program to Calculate Electron Paramagnetic Resonance Hyperfine Couplings in Semiquinone Anion Radicals” Alice Haddy (2001) Journal of Chemical Education 78, 1206-1208
“EPR Characterization of the Effects of Azide on Photosystem II” Alice Haddy, R. Allen Kimel, and Rebecca Thomas (2000) Photosynthesis Research 63, 35-45
“Azide as a Competitor of Chloride in Oxygen Evolution by Photosystem II” Alice Haddy, J. Andrew Hatchell, R. Allen Kimel and R. Thomas (1999) Biochemistry 38, 6104-6110